The project seeks to determine if prior exposure to mild ischemia, such as occurs during Transient Ischemic Attack (TIA), alters the brain's response to subsequent stroke by serving as a preconditioning stimulus and triggering neuroprotective mechanisms. Specifically, we will test the hypothesis that experimental TIA reduces damage from subsequent stroke by a mechanism that involves upregulation of cytochrome P450 2C11, a P450 AA epoxygenase expressed in astrocytes, which catalyzes the conversion of arachidonic acid (AA) to biologically active epoxyeicosatrienoic acids (EETs). The Specific Aims of the project are: 1) To determine if experimental TIA protects brain from subsequent stroke by EETs-mediated mechanism, 2) To determine if TIA-enhanced EETs production is due to upregulation in astrocytes of the biosynthetic enzyme P450 2C11, 3) To determine if genetic and pharmacological manipulations of EETs synthetic and breakdown pathways mimic the protection acquired by TIA, and 4) To determine if ischemic tolerance in TIA-preconditioned brain is due to EETs-mediated activation of phosphoinositide 3-kinase (P13-K) /Akt cell survival signaling cascade in neurons. To simulate TIA, three 10-minute periods of middle cerebral artery occlusion (MCAO) will be induced in rats and mice; three days later, a more severe 2-hour ischemia is produced and infarct size is measured at 1 and 7 days to assess short- and long term tissue outcome after stroke. The level of endogenous EETs in brain will be manipulated using pharmacological and genetic tools and measured using in-vivo microdialysis. Expression of P450 2C11 will be examined using RNase protection assay, Western blotting, immunohistochemistry and in-situ hybridization. Western blotting with anti-phospho-Akt antibody will assess Akt activation and the importance of this pathway to ischemic tolerance will be evaluated using specific P13-K/Akt inhibitors. The studies explore a potential endogenous mechanism of protection from cerebral ischemia, which can be targeted as a novel strategy for stroke injury prevention and treatment.